The cyclin-dependent kinase inhibitor p27 is required for an effective cell cycle arrest in vivo. This arrest is relieved by degradation of p27 via the ubiquitin-dependent proteolysis system. Depletion of the p27 tumor suppressor resulting form hyperproteolysis is a hallmark of many advanced carcinomas that correlates with decreased survival. P27 degradation is mediated by SCFSKP2-dependent polyubiquitylation and SKP2 overexpression is sufficient to cause ectopic p27 degradation. Hyperactivation of the SKP2-mediated proteolysis pathway therefore appears to be the main mechanism of p27 downregulation in carcinomas and a good target for therapeutic intervention. Small molecules able to interfere with SKP2-dependent p27 degradation are predicted to [unreadable] restore p27 expression in carcinomas thus inhibiting their unrestrained growth. This prediction constitutes the principal hypothesis of this grant proposal. In preliminary work, 7368 compounds were screened in a 384-well plate format for their ability to upregulate p27 in prostate cancer cells overexpressing SKP2. 54 compounds (named SMIPs) were identified and verified, which were effective with a z score of >3 - 5. While this work has provided proof-of-principle for the feasibility of [unreadable] identifying SMIPs, the screening assay relied on detection of p27 by immunofluorescence and is therefore not suitable for high-throughput screening. In this application, an optimized, cell-based "mix-and-measure" assay for the identification of SMIPs is proposed. The assay will rely on stable expression of a YFPp27 fusion protein in HeLa cells. The addition of compounds that upregulate YFP-p27 will result in an increased fluorescence signal that will be measured by cellular imaging. The assay will also allow real-time measurement of SMIP activity. If validated, the assay will be used to perform a SMIP screen at the San Diego Center for Chemical Genomics, one of the Molecular Library Screening Centers in the NIH Roadmap Network. [unreadable] [unreadable] [unreadable]